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International audience; The pathological features of Whipple endocarditis, which is caused by Tropheryma whipplei, were histologically evaluated in cardiac valves from 5 patients. We used quantitative image analysis to compare the valvular fibrosis, calcifications, vegetations, inflammation, and vascularization due to Whipple endocarditis with those due to non-Whipple endocarditis and degenerative valves. We also studied the presence of T. whipplei in valves by immunohistochemical analysis, culture, and polymerase chain reaction (PCR). In histologic analysis, Whipple endocarditis was characterized by significant fibrosis, a lack of calcifications, slight inflammation and vascularization, and vegetations of intermediate size. Inflammatory infiltrates consisted mainly of foamy macrophages and lymphocytes. We found that the detection of T. whipplei in cardiac valves, by immunohistochemical analysis, was correlated with the detection of the bacterium by culture and PCR. We report, for the first time, the immunodetection of T. whipplei in a surgically removed arterial embolus. Pathological and immunohistologic analyses may contribute to the diagnosis of Whipple endocarditis.

Whipple disease (WD) is a systemic disorder caused by the bacterium Tropheryma whipplei. Since the recognition of a bacterial etiology in 1961, many attempts have been made to cultivate this bacterium in vitro. It was eventually isolated, in 2000, from an infected heart valve, in coculture with human fibroblasts. Here we report the isolation of 2 new strains of T. whipplei from cerebrospinal fluid (CSF) of 2 patients with intestinal WD but no neurological signs or symptoms. One culture-positive specimen was obtained before treatment; the other was obtained 12 months after discontinuation of therapy, at a time of intestinal remission. In both cases, 15 passages of the cultures were completed over 17 months. Bacterial growth was measured by quantitative polymerase chain reaction, which suggested a generation time of 4 days. Staining with YO-PRO nucleic-acid dye showed characteristic rod-shaped bacteria arranged in chains. Fluorescent in situ hybridization with a T. whipplei-specific oligonucleotide probe, a broad-range bacterial probe, and a nonspecific nucleic-acid stain indicated that all visible bacteria were T. whipplei. Scanning electron microscopy and transmission electron microscopy showed both intracellular and extracellular bacteria. This first isolation of T. whipplei from CSF provides clear evidence of viable bacteria in the central nervous system in individuals with WD, even after prolonged antibiotic therapy.

Whipple disease (WD) is a systemic disorder caused by the bacterium Tropheryma whipplei. Since the recognition of a bacterial etiology in 1961, many attempts have been made to cultivate this bacterium in vitro. It was eventually isolated, in 2000, from an infected heart valve, in coculture with human fibroblasts. Here we report the isolation of 2 new strains of T. whipplei from cerebrospinal fluid (CSF) of 2 patients with intestinal WD but no neurological signs or symptoms. One culture-positive specimen was obtained before treatment; the other was obtained 12 months after discontinuation of therapy, at a time of intestinal remission. In both cases, 15 passages of the cultures were completed over 17 months. Bacterial growth was measured by quantitative polymerase chain reaction, which suggested a generation time of 4 days. Staining with YO-PRO nucleic-acid dye showed characteristic rod-shaped bacteria arranged in chains. Fluorescent in situ hybridization with a T. whipplei-specific oligonucleotide probe, a broad-range bacterial probe, and a nonspecific nucleic-acid stain indicated that all visible bacteria were T. whipplei. Scanning electron microscopy and transmission electron microscopy showed both intracellular and extracellular bacteria. This first isolation of T. whipplei from CSF provides clear evidence of viable bacteria in the central nervous system in individuals with WD, even after prolonged antibiotic therapy.

Whipple's disease is caused by a cultivation-resistant bacterium, Tropheryma whippelii. Ultrastructural studies of intestinal biopsy specimens from patients with Whipple's disease have shown that intracellular and extracellular bacteria are present, but the preferred site of growth is unknown. Tissue sections from 8 patients with Whipple's disease and from 19 healthy control subjects were analyzed by use of fluorescence in situ hybridization and laser scanning confocal microscopy, to determine the location of rRNA that would indicate the presence of metabolically active bacteria. T. whippelii rRNA was most prevalent near the tips of intestinal villi, in the lamina propria, just basal to epithelial cells. Most of the bacterial rRNA signal appeared to be located between cells and did not colocalize with the human intracellular protein vimentin. The location of bacterial rRNA in tissues from patients with Whipple's disease provides evidence that bacteria are growing outside cells and suggests that T. whippelii is not an obligate intracellular pathogen.

Background. Whipple disease is a chronic infection caused by Tropheryma whipplei. Trimethoprim-sulfamethoxazole is recommended for treatment of Whipple disease but is associated with treatment failure. T. whipplei is resistant in vitro to trimethoprim, because the gene targeted by this agent is missing. Methods. A patient experienced clinical failure during treatment with trimethoprim-sulfamethoxazole. The gene encoding the enzyme putatively believed to be dihydropteroate synthase (DHPS), the target of sulfamethoxazole, was amplified and sequenced for 20 T. whipplei strains from our laboratory and for isolates recovered from a case patient at the time of diagnosis and the time of treatment failure. An Escherichia coli knockout strain for this gene was complemented with the sequences from a susceptible strain and from isolates recovered from the case patient. Susceptibilities of complemented E. coli to sulfamethoxazole were tested. Results. The target gene was identified among genes encoding a unique trifunctional enzyme in which DHPS is combined with the 2 preceding enzymes of the folate biosynthesis pathway. Changes in the amino acid sequence of putative DHPS were detected in the case patient. Gene complementation showed that the gene encoding putative DHPS restored the folate biosynthesis pathway and susceptibility to sulfamethoxazole, whereas the mutated sequence was associated with sulfamethoxazole resistance. Conclusions. Antibiotic susceptibility of fastidious bacteria such as T. whipplei can be evaluated by means of gene complementation techniques. Mutations in the target gene of sulfamethoxazole appear during treatment.

Background. The reservoir of the agent of Whipple disease is unknown. Asymptomatic carriage of Tropheryma whipplei in human stool and saliva is controversial. Methods. Stools and saliva specimens from 231 workers at a sewage treatment facility and from 10 patients with Whipple disease, stool specimens from 102 healthy people, and stool specimens from 127 monkeys or apes were tested for T. whipplei DNA by a quantitative real-time polymerase chain reaction with probe detection. Genotyping and culture of T. whipplei-positive samples were performed. Results. Asymptomatic carriage in stool was found in humans (ranging from a prevalence of 4% in the control group to 12% among a subgroup of sewer workers) but not in monkeys and apes. The T. whipplei load in stool was significantly lower in carriers than in patients with Whipple disease (P

We tested 53 fixed duodenal biopsy samples from 25 patients with Whipple disease for the presence Giardia and 6 fresh duodenal biopsy samples for Giardia duodenalis DNA by polymerase chain reaction (PCR). We also used histological examination and PCR assay to determine the prevalence of G. duodenalis in duodenal biopsy samples from 150 control patients without Whipple disease. Three of 25 patients with Whipple disease had histological evidence of giardiasis, whereas only 1 of 150 control patients was affected (P

Whipple's disease is a systemic illness caused by a specific agent. Despite recognition of bacteria in lesions, efforts to isolate the causative agent remained futile. A novel strategy was devised to culture Whipple bacilli in deactivated mononuclear phagocytes. Infected tissue was inoculated into human phagocytes deactivated with interleukin (IL)-4, IL-10, and dexamethasone. Within 8-10 days, diastase-resistant periodic acid-Schiff-positive inclusions appeared, corresponding to intact and degenerating bacteria shown to be Tropheryma whippelii by electron microscopy and molecular analyses. T. whippelii was passaged several times in deactivated monocytes and a monoblastic cell line. Time-kinetics growth studies and comparative polymerase chain reaction analysis documented multiplication of T. whippelii in deactivated macrophages. Complementary studies showed that IL-4 rendered phagocytes permissive for T. whippelii, a strong indication that host factors contribute to the pathogenesis of Whipple's disease. The propagation of T. whippelii will permit microbiologic, immunologic, seroepidemiologic, and therapeutic studies of this pathogen.

Clinicians have long been aware of human diseases that are associated with visible but uncultured microorganisms. Without the ability to cultivate these organisms, they have remained unidentified. Environmental microbiologists have also discovered on the basis of recent advances in the field of molecular phylogeny that culture-based methods for detecting microorganisms are biased and insensitive. A culture-independent experimental approach is described for the identification of microbial pathogens. This approach incorporates fundamental aspects of 16S rRNA-based molecular phylogeny as well as nucleic acid amplification technology. From its application to Whipple's disease, one can speculate as to the potential insights a highly sensitive, culture-independent method may provide into the diversity and natural ecology of human microbial pathogens.